Field of the Invention
The present invention relates to the fields of laser ablation and mass spectrometry. The present invention also relates specifically to methods and devices for plume capture of laser ablated samples for mass spectrometry and spectroscopy.
Related Art
Determining the chemical composition of complex biological systems, such as tissues, biofilms, and bacterial colonies, presents a daunting analytical challenge. The composition of such samples are typically heterogeneous and dynamic, changing both in time and in response to varying environmental conditions, requiring methods of analysis that can provide chemical information with both high spatial and temporal resolution. The ability to measure and image the chemical composition of biological samples under native conditions and with minimal modification/preparation is important to advancing our understanding of processes, such as cell differentiation,1-3 photosynthesis4-5 and cellular metabolism.6-9.
There are many microanalysis techniques for characterizing the chemical composition of biological samples, including NMR/MRI,10-11 visible microscopy, infrared spectromicroscopy,1, 6-8 Raman imaging,12-13 fluorescence-tagging and imaging of molecules,14-15 and imaging mass spectrometry.16-30 (See References 14-20), Many of these techniques can provide high spatial resolution and are non-destructive, but often do not provide unambiguous chemical information. Fluorescence-tagging of molecules can provide images with both very high spatial resolution (˜1-200 nm) and precise molecular specificity by using antibodies to target specific molecules. However, only a few components can be imaged simultaneously through the use of fluorophores with different emission wavelengths, and the procedure for tagging molecules with fluorophores often requires extensive sample preparation. Imaging mass spectrometry provides chemical information with excellent molecular specificity and can be used to generate images for up to thousands of compounds measured simultaneously.30 Mass spectrometry can also be combined with other imaging techniques to provide multimodal imaging analysis.31-34 Unlike many optical methods, mass spectrometry is a destructive technique; molecules must be removed from the sample and be ionized to be detected.
The most widely-used mass spectrometry imaging techniques are matrix-assisted laser desorption ionization (MALDI)16-19, 22-23, 30_ and secondary ion mass spectrometry (SIMS).16-21 Conventional MALDI and SIMS are often used to generate chemical images for fixed tissue samples. For both techniques, ions are generated under vacuum and are subsequently mass analyzed. Because vacuum is required for these techniques, neither is suitable for the analysis of living systems. MALDI typically involves the application of an external and usually denaturing matrix chemical which absorbs the energy from a laser resulting in ablation and ionization. With SIMS, secondary ions are sputtered off a surface with a beam of primary ions, such as Cs+ or polyatomic Aun+ clusters. Chemical images can be obtained with very high spatial resolution (˜100 nm),20 but the sensitivity for high mass ions (m/z>1000) can be poor due to low secondary ion yields.18-21, 35.
Many techniques for imaging mass spectrometry at ambient pressure have been recently introduced. Cooks and co-workers introduced a now widely used method, desorption electrospray ionization (DESI), in 2004.28 With DESI, a plume of charged solvent droplets generated by electrospray is directed at a sample surface and the charged droplets desorb and ionize chemical components from the sample surface. Many other ambient imaging mass spectrometry techniques have subsequently been developed. With nano-DESI36-37 and liquid micro-junction surface sampling probe (LMJ-SSP),38-40 solvent is flowed over a small area of sample and then carried to an ESI emitter. Numerous methods use laser light to select spatially resolved areas for mass analysis. These methods include electrospray-assisted laser desorption ionization (ELDI),41-42 atmospheric pressure infrared MALDI (AP IR-MALDI),43-44_matrix-assisted laser desorption electrospray ionization (MALDESI),29, 45-46_laser ablation electrospray ionization (LAESI),26, 47 and IR laser ablation sample transfer (LAST).24-25, 27, 48-49 
Methods that use IR-laser ablation can take advantage of the water naturally present in biological samples as a matrix to absorb IR radiation. The IR laser pulse produces surface evaporation, phase explosion (explosive boiling) of water and the secondary ejection of sample material into a plume of fine droplets.50-51 The ejected sample material consists of mostly neutral droplets/particles which can be ionized by intersection with an electrospray plume (ELDI, LAESI, MALDESI) or can be captured in solvent (LAST) for subsequent ionization by electrospray. With AP IR-MALDI, the fraction of molecules directly ionized by the laser ablation process are introduced into the mass spectrometer. The energy deposited into solute molecules by the laser ablation process has been studied using thermometer ions with well known fragmentation energies (i.e. benzyl-substituted benzylpyridinium salts), and with peptides (i.e. bradykinin, substance P). Water/methanol solutions of these compounds were air-dried onto plant leaves and laser ablated with energy densities of up to 15 J/cm2. Based on comparison of the fragmentation product intensities measured for LAESI and ESI experiments under varying fragmentation conditions, the infrared laser ablation was reported to have little effect on the internal energy distribution of the resulting ions for laser energy densities up to 15 J/cm2.51 
High transfer efficiency is especially important for the analysis of biological samples due to low concentrations of some molecular species within the highly complex mixtures of biochemicals from living cells. The transfer efficiency from a 1 mM solution of angiotensin II to flowing solvent by backside geometry laser ablation was reported to be 2%.25 This value was estimated by comparing the signal obtained from the laser ablation of a known quantity of angiotensin II with the signal obtained from direct electrospray ionization of an angiotensin II standard solution. LAESI, in which the ablation plume expands into a flow of highly charged solvent droplets produced by electrospray, is reported to be “characterized by significant sample losses and low ionization efficiencies.”26 Vertes and co-workers reported that the transfer efficiency of LAESI was improved by the use of a capillary to confine the sample and to direct the radial expansion of the ablation plume, guiding more material directly into the electrospray flow and described in Stolee, J. A.; Vertes, A., Toward Single-Cell Analysis by Plume Collimation in Laser Ablation Electrospray Ionization Mass Spectrometry. Anal. Chem. 2013, 85, 3592-3598, hereby incorporated by reference.26 